The Role Of Autophagy In The Ovarian Follicles During Ovarian Vitrification And Transplantation

Objective The survival rate of cancer patient was significantly increased with the enhanced medical technology,accompanying with the increased fertility preservation of cancer patient.Ovarian cryopreservation by vitrification and transplantation are main methods to preserve fertility for cancer patient,especially for young women who suffered with cancer.But,the dysfunction of oocyte and vacuolization of ovarian follicle are caused by the transplantation of vitrified/warmed ovaries.Therefore,the preservation of ovarian follicle,especially primordial follicle is very important for ovarian grafts.Previous studies suggested that the survive of ovarian follicle was preserved by autophagy through anti-cryoinjury,additionally,the type II program cell death,autophay involved in the ovarian follicle atresia,whether autophagy mediated cell death was initiated during ovarian vitrification and transplantation is fully unknown.So,21-days-old ICR female mouse ovaries were used to this work,the process of ovarian vitrification and transplantation was intervened by autophagy inhibitor 3-Methyladenine and Bafilomycin A1,activator rapamycin,and the role and mechanism of autophagy in the ovarian vitrification and transplantation are explored.MethodsThe first experiment:The study of the role of autophagy in ovarian cryopreservation by vitrification.The mouse ovaries were collected and divided into five groups: Control group(fresh ovaries),the ovaries were done without cryopreservation(cryoprotectant group),vitrified groups,autophagy inhibitor intervened group(3-MA and Baf1),autophagy activator groups(Rapa).The effect of autophagy on vitrified ovaries and the optimal intervene concentration of autophagy inhibitor and activator were explored with histology and ovarian follicle count;the autophagy molecular marker LC3 was detected by western blot;the apoptosis of ovarian follicle was detected with TUNEL and staining with cleaved caspase-3.The autophagosome was detected by electronic scanner microscope.Through these methods,the role of autophagy was uncovered in the ovarian vitrification.The second experiment: The study of the role autophagy in the ovarian transplantation.The protocol contains two groups.Group one: fresh ovaries were transplanted into receptor;Group two: vitrified/warmed ovaries were transplanted into receptor,and the 6-8 weeks old female mice were used as receptor.The group one and group two were divided into three sub-groups respectively: fresh ovary group or vitrified/warmed ovary group(only saline was administreted with the receptor 48 h,24 h before the transplatation);3.75 mg/kg 3-MA treated group(3.75 mg/kg 3-MA was administreted with the receptor 48 h,24 h and 30 min before the transplatation),7.5 mg/kg 3-MA treated group(7.5 mg/kg 3-MA was administreted with the receptor 48 h,24 h and 30 min before the transplatation).Results Results of the first experiment.(1)Observation of ovarian morphological histology and follicle count: The ovarian morphological histology results suggested that the ovarian follicles in the fresh samples were all intact,while the ovarian follicles in the vitrified/warmed,3-MA,Baf1,Rapa and CPA groups were scattered with shrinkage of some oocytes.The number of ovarian follicles was counted,and results suggested that the number of primordial follicles in the 3-MA,CPA and Rapa groups was significantly decreased compared with fresh and vitrification groups(p<0.05).The fresh and vitrification groups had no significant differences,while the number of primary follicles was not significantly different between the groups.The number of antral follicles in the 3-MA,CPA and Rapa groups was significantly decreased compared with fresh group(p<0.05),and there were no significant differences in the fresh,vitrification and Baf1 groups.The number of atretic follicles in 3-MA,CPA,Baf1 and Rapa groups was significantly increased compared with fresh group(p<0.05),but the number of atretic follicles in group of vitrification was increased compared with fresh groups,although without significant difference.(2)Autophagosome observation with electron microscope: To confirm the autophagy in the ovarian vitrification,the fresh ovary(Fig.3A),vitrified/warmed ovary(Fig.3B),CPA ovary(Fig.3C)and 3-MA-treated(Fig.3D)ovary were detected with electron microscope.The the presence of autophagosome was detected in the four groups,suggesting that the autophagy was involved in the process of ovarian vitrification.The structure of organelles,especially the mitochondria,in the vitrified/warmed ovary and fresh ovary,was more intact,while it was scattered in the CPA ovary and 3-MA ovaries.(3)Apoptotic rate of ovaries: The TUNEL results suggested that the apoptotic signal was detected in all ovaries,while apoptotic signal was primarily localized in the granulosa cell and oocyte(Fig.4).The apoptotic rate was analyzed,and results suggested that the apoptotic rate was significantly increased in the vitrification,CPA,3-MA,Baf1 and Rapa groups compared with fresh group(p<0.05),although there were no significant differences in the vitrification,3-MA,Baf1 and Rapa groups(Fig.6a).Furthermore,the apoptotic marker cleaved caspase-3 was also checked by immunofluorescence(Fig.5),and the results were consistent with the TUNEL,which demonstrated that the expression of cleaved caspase-3 was significantly increased in the vitrification,CPA,3-MA and Baf1 groups compared with fresh group(p<0.05)(Fig.6b).This result was further supported by western blot analysis(Fig.6c),in which the expression of cleaved caspase-3 was significantly increased in the vitrification,CPA,3-MA,Baf1 and Rapa groups compared with fresh group(p<0.05)(Fig.6d).In addition,the expression of autophagy molecular marker LC3 was checked by western blotting(Fig.6c),and the expression of autophagy levels in the vitrification group was significantly higher than in the fresh groups(p<0.05),suggesting that the autophagy was activated in the process of ovarian vitrification.Results of the second experiment: The histological results suggested that the ovarian morphology in the fresh ovary transplantation group was relatively intact,but the loss a nd atresia of ovarian follicle was happened,especially more primordial follicle was lost,and the stromal cell was damaged,and this situation was aggravated in the 7 d and 42 d after transplantation.In vitrified/warmed ovary transplantation groups,compared with ovarian grafts from vitrified/warmed ovary with 3-MA treated receptor,the losses of ovarian follicle and fibrosis of ovarian grafts in control groups was remarkably increased,and there is no primordial follicle in control groups,but the primordial follicle was detected in the 3-MA treated groups.In addition,the angiopoiesis was detected with staining the vascular endothelium molecular marker CD31 and CD34,the number of micro-vessel was count and results suggested that the number of micro-vessel of 3-MA groups was significantly increased compared with control groups(P<0.05).Conclusions(1)The autophagy was increased in the ovarian vitrification,and the ovarian follicle,especially of primordial follicle was preserved by autophagy through anti-cryoinjury.(2)The survival of ovarian follicle,especially primordial follicle was increased,and angiopoiesis was increased during the process of ovarian transplantation with autophagy inhibitor 3-MA treatment.